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Abstract
Abstract Tungsten or vanadium in a nutrient medium containing nitrate, but not ammonia, as the sole nitrogen source inhibited the formation of assimilatory NADPH-nitrate reductase (but not nitrate-inducible NADPH-cytochrome c reductase) and growth of wild type Neurospora crassa. Inhibition was prevented by high levels of molybdate in the nutrient medium but was not reversed by adding molybdate to subsequently prepared cell-free enzyme preparations. Neither tungsten nor vanadium was inhibitory when added to partially purified wild type Neurospora NADPH-nitrate reductase. Cell-free preparations of uninduced wild type grown in increasing concentrations of tungsten or vanadium showed progressive inhibition of ability to assemble nitrate reductase in vitro (with extracts of nitrate-induced Neurospora mutant nit-1) which was partially or entirely restored by subsequent addition of molybdate to the extracts of the tunsgten- or vanadium-grown uninduced wild type. Tungsten or vanadium added to acid-treated bovine milk xanthine oxidase or to extracts of uninduced wild type also inhibited in vitro enzyme assembly, with added molybdate partially preventing inhibition. The tungsten or vanadium analogue of nitrate reductase formed in vivo or by in vitro assembly is considerably more labile than its molybdenum counterpart as indicated by the complete loss of 185W or 48V from the enzyme as a result of trichloroacetic acid precipitation or heat treatment. Sucrose density gradient profiles of the Neurospora NADPH-nitrate reductase formed in vitro with 185W- or 48V-grown uninduced wild type or induced nit-1 showed, as in the case of 99Mo, that uninduced wild type and not nit-1 can be the source of tungsten or vanadium in the in vitro assembly of the enzyme. Exogenous 185W added to cell-free extracts of uninduced wild type or acid treated bovine milk xanthine oxidase prior to in vitro assembly of nitrate reductase using nit-1 failed to appear in the subsequently formed enzyme, whereas 48V experienced a small but consistent incorporation only with acid-treated xanthine oxidase. There was no in vitro exchange or incorporation of 185W or 48V into the already formed wild type enzyme. The data implicate tungsten and vanadium as competitive inhibitors of molybdenum in nitrate reductase structure and function.
Highlights
MethodsCulture Methods, Substrates, Cofactors, and Other SubstancesNeurospora crassa wild type STA4 and the nitrate reductase mu-
Tungsten or vanadium in a nutrient medium containing nitrate, but not ammonia, as the sole nitrogen source inhibited the formation of assimilatory NADPH-nitrate reductase
Tungsten or vanadium added to acidtreated bovine milk xanthine oxidase or to extracts of uninduced wild type inhibited in vitro enzyme assembly, with added molybdate partially preventing inhibition
Summary
Culture Methods, Substrates, Cofactors, and Other SubstancesNeurospora crassa wild type STA4 and the nitrate reductase mu-. Vivo labeling of iVeurospora with each of the radioactive isotopes was generally attained by growth in Fries basal medium (from which molybdenum had been omitted) supplemented with either (a) ammonium chloride as the sole nitrogen source and the radioactive salt as in the case of uninduced wild type, or (b) ammonium chloride as the sole nitrogen source and the radioactive salt, followed by induction in Fries basal medium containing sodium nitrate as the sole nitrogen source and the radioactive salt, as in the case of induced wild type and induced ?Lit-1. The levels of radioactive salts added to the growth medium and to the induction medium were 150 rrCi/lOO ml of medium for K,*85W0d or 48V0,C1
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